Immature CD8 Single-Positive Thymocytes Are a Molecularly Distinct Subpopulation, Selectively Dependent on BRD4 for Their Differentiation

Abstract

T cell differentiation in the thymus proceeds in an ordered sequence of developmental events characterized by variable expression of CD4 and CD8 coreceptors. Here, we report that immature single-positive (ISP) thymocytes are molecularly distinct from all other T cell populations in the thymus in their expression of a gene profile that is dependent on the transcription factor BRD4. Conditional deletion of BRD4 at various stages of thymic differentiation reveals that BRD4 selectively regulates the further differentiation of ISPs by targeting cell cycle and metabolic pathways, but it does not affect the extensive proliferation that results in the generation of ISPs. These studies lead to the conclusion that the ISP subpopulation is not a hybrid transitional state but a molecularly distinct subpopulation that is selectively dependent on BRD4.

The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

“…As summarized in Figure 1C, splicing patterns were affected by BRD4 deletion in each of the subpopulations, with both increases and decreases in the different splicing events (Figure S1A). Consistent with BRD4 preferentially affecting the maturation of the ISP subpopulation (Gegonne et al, 2018), the largest differences occurred in this population. Namely, within the ISP population, there were a total of 241 differences in splicing events among expressed genes between the BRD4-deficient ISPs and the WT.…”

“…The SE was the predominant form affected by BRD4 deletion, though all of the forms were affected to some extent (Figures 1A and S1A). Of note, there were no significant effects of BRD4 deletion on the level of any of the splicing factors (Gegonne et al, 2018). These data demonstrate that BRD4 contributes to the splicing of endogenous genes in vivo .…”

“…BRD4 is expressed in all five subpopulations, at approximately equal levels (Figure S1B). Deletion of BRD4 at the DN stage causes a selective defect in the RNA expression profile in the ISP thymocytes, targeting the cell cycle and metabolic pathways and severely inhibiting their further differentiation (Gegonne et al, 2018). In contrast, BRD4 had only a modest effect on the RNA expression profiles of the remaining thymocyte subpopulations, while their differentiation was noticeably affected.…”

E-protein–regulated expression of CXCR4 adheres preselection thymocytes to the thymic cortex

“…We began assessing a possible role for E-protein transcription factors in thymic retention of preselection thymocytes by conditionally deleting Tcf3 and Tcf12 genes, which encode E2A and HEB proteins, respectively. Conditional deletion of floxed Tcf3 and Tcf12 genes was accomplished with either CD4-Cre recombinase, which deletes at the late DN/immature SP (ISP) thymocyte stage (E2Δ ISP ; Lee et al, 2001 ; Gegonne et al, 2018 ), or E8 III -Cre recombinase, which deletes at the DP thymocyte stage (E2Δ DP ; Park et al, 2010 ). We found that conditional deletion of E-proteins in germline ZAP KO mice caused both TCR − CD8 + and TCR + CD8 + T cells to appear in the periphery even though their precursors could not possibly have been TCR-signaled in the thymus because of absent ZAP70 ( Fig.…”

“…1 C ). Because the Cre-recombinase was expressed in DN/ISP thymocytes in E2Δ ISP mice ( Lee et al, 2001 ; Gegonne et al, 2018 ) and was expressed in DP thymocytes in E2Δ DP mice ( Park et al, 2010 ), we considered that the TCR-unsignaled TCR − CD8 + thymocyte populations in these mice might be at different stages of thymocyte development, i.e., before and after DP. If so, generation of TCR − CD8 + thymocytes in E2Δ ISP and E2Δ DP mice would have different cytokine requirements, since in vivo cytokine signals induce the Runx-family transcription factor Runx3d, which up-regulates CD8 expression only on post-DP thymocytes ( Park et al, 2010 ; McCaughtry et al, 2012 ; Etzensperger et al, 2017 ), but cytokine signals are not required to induce the Runx-family transcription factor Runx1, which up-regulates CD8 expression on pre-DP thymocytes ( Egawa et al, 2007 ; Etzensperger et al, 2017 ).…”